The long term goal of the project is to understand the mechanism of oxidative phosphorylation with regard to the molecular mechanism of ATp formation on the mitochondrial ATPase (F1) and the molecular mechanism of coupling between the energy site in the mitochondrial membrane and the ATP synthesizing apparatus. This project considers that during oxidative phosphorylation ATP is formed in catalytic sites on F1 from bound ADP and Pi with virtually zero change in free energy and that the major requirement for energy in oxidative phosphorylation is for the release of product ATP from high affinity catalytic sites. The project will examine the reaction mechanism of the ATPase in soluble and membrane-bound forms. It will determine whether the action of the catalytic sites is simultaneous or sequential, it will study changes in the enzyme subsequent to substrate binding that would be indicative of conformational change and it will study pH-dependent changes in the equilibrium constants of the reaction mechanism, particularly changes in product release, as an approach to the nature of the interactions of the ATPase with the coupling mechanism. A study of the catalytic site cooperativity and of reversible Pi and ATP-dependent activation of the enzyme are expected to provide further insight into the reacton mechanism of the enzyme. The methodological approaches to be employed include quenched flow measurements for determining equilibrium constants in the reaction mechanism, stop flow techniques to search for intermediates in the initial steps of ATP hydrolysis and the centrifuge column.